1. Introduction
The following description includes information that may be useful in understanding the present invention. It is not an admission that any such information is prior art, or relevant, to the presently claimed inventions, or that any publication or patent specifically or implicitly referenced is prior art.
2. Background
The immunologic arts have advanced markedly in recent years. The complexity of the science explaining aspects of the field is immense. Set forth below is a discussion concerning known aspects of various elements involved in immunogenic responses and concepts in the art that are related to the invention disclosed herein.
A. T Cells
T lymphocytes (i.e., T cells) are part of the immune system, which defends the body against bacterial, viral, and protozoal infection, as well as molecules that contain epitopes recognized as non-self (including aberrant forms of molecules that occur naturally in the body). The recognition of non-self molecules as well as the destruction of infectious agents carrying non-self antigens is a function of T cells, which, along with certain B lymphocytes (i.e., B cells), provide for the cell-mediated immune responses of adaptive immunity.
Infecting pathogens are generally accessible to extracellular antibodies found in the blood and the extracellular spaces. However, some infecting agents, including all viruses, replicate inside cells where they are not exposed to, and thus cannot be detected by, extracellular antibodies. In order for these foreign agents to be accessible to the cell-mediated immune response, cells harboring such pathogens must either “express” antigenic motifs of the infecting agents on the surface of the cells or the antigenic motifs must be shed from the cells, e.g., by cell death, to be accessible to and subsequently expressed on the cell membrane of phagocytic antigen presenting cells (“APCs”) that participate in cell-mediated immune processes.
Antigens derived from replicating viruses, for example, are displayed on the surface of infected cells where they may be recognized by “cytotoxic” T cells or other surveillance cells such APCs, e.g., dendritic cells. T cells may then respond to the infection by recognizing the viral antigens and then killing the infected cell. The actions of such cytotoxic T cells depend upon direct interaction between the antigenic motif of the infecting agent expressed on the surface of the infected cells and T cell receptors specific for the motif.
Although T cells are important in responding to intracellular infections, some foreign agents evade such responses because they replicate in the vesicles of macrophages, as occurs with Mycobacterium tuberculosis, the pathogen that causes tuberculosis. Whereas bacteria that enter macrophages are usually destroyed in lysosomes (which contain a variety of enzymes and bactericidal substances), infectious agents such as M. tuberculosis may survive because the vesicles they occupy may not fuse with macrophage lysosomes. The immune system fights such agents using a second type of T cell, known as a “T helper cell,” which helps to activate macrophages and induce the fusion of lysosomes with vesicles containing the infectious agents. The T helper cells also bring about the stimulation of other immune mechanisms of the phagocyte. T helper cells may further be involved in initiating and/or sustaining the immune system's release of soluble factors that attract macrophages and other professional APCs to the site of infection.
Additionally, specialized T helper cells play an important role in the destruction of extracellular pathogens by interacting with B cells. Depending on the type of infection being controlled, participating T helper cells may have an inflammatory, or Th1-like phenotype, or a suppressive, Th2-like phenotype. Other T helper cell types may also exist, including Treg and Th3 cells. In the case of cytotoxic T cells and Th1 cells, fragments of non-self proteins (i.e., processed antigens) are recognized on the surface of the target cell (such as an infected cell). Th2 cells, on the other hand, recognize and interact with antigen presented by professional antigen presenting cells such as dendritic cells and B cells. Dendritic cells non-specifically internalize foreign antigens while B cells may bind and internalize foreign antigens via their antigen-specific cell surface immunoglobulin. In any case, T cells recognize their targets by detecting non-self antigenic motifs (e.g., peptide fragments derived from, for example, a bacterium or virus) that are expressed either on infected cells or other immune cells, e.g., phagocytic APCs.
The actions of T cells depend on their ability to recognize antigenic motifs on cells (such as cells harboring pathogens or that have internalized pathogen-derived products). T cells recognize peptide fragments (e.g., pathogen-derived proteins) in the form of complexes between such peptides and MHC (major histocompatibility complex) molecules that are expressed on the surface of antigen presenting cells.
B. T Cell Receptors
T cell receptors (TCRs) are closely related to antibody molecules in structure, and they are involved in antigen binding although, unlike antibodies, they do not recognize free antigen; instead, they bind antigen fragments which are bound and presented by antigen-presenting molecules. An important group of antigen-presenting molecules are the MHC class I and class II molecules that present antigenic peptides and protein fragments to T cells. Other antigen presenting molecules have also been identified, including CD1, which present lipid and glycolipid antigens to T cells.
Variability in the antigen binding site of a TCR is created in a fashion similar to the antigen binding site of antibodies, and also provides specificity for a vast number of different antigens. Diversity occurs in the complementarity determining regions (CDRs) in the N-terminal domains of the disulfide-linked alpha (α) and beta (β), or gamma (γ) and delta (δ), polypeptides of the TCR. The CDR loops are clustered together to form an MHC-antigen-binding site analogous to the antigen-binding site of antibodies, although in TCRs, the various chains each contain two additional hypervariable loops as compared to antibodies. TCR diversity for specific antigens is also directly related to the MHC molecule on the APC's surface to which the antigen is bound and presented to the TCR.
C. MHC Molecules
In general, T cell responses to “non-self” peptide motifs depend on the interactions of the T cells with other cells that express of otherwise contain the proteins recognized as non-self. The molecules that associate with these peptide or antigen fragments and present them to T cells are membrane glycoproteins encoded by a cluster of genes that comprise the MHC. In general, the term MHC is used to describe generally the molecules in the mammalian immune system involved in the presentation of antigenic motifs to T cells. Here, MHC means any major histocompatibility complex class I or class II protein from any mammalian organism, including a human, mouse, rat, horse, pig, dog, cat, or sheep, that present antigens to T cells. Such molecules include full-length MHC molecules or subunits thereof, and further include MHC-encoded antigen-presenting glycoproteins having the capacity to bind a antigenic peptides. MHC proteins may comprise an amino acid sequence that is expressed and purified from natural sources, or by any synthetic techniques such as recombinant expression of a desired gene, or group of genes, in prokaryotic or eukaryotic systems, which can, for example, result in different patterns of glycosylation. The MHC sequence may comprise a natural or modified amino acid sequence, including those that are truncated at either or both the C- or N-terminus and/or include one or more amino acid insertions, deletions, or substitutions in a naturally occurring MHC sequence.
The two types of MHC molecules, i.e., MHC class I and MHC class II molecules, deliver peptides from different sources (class I MHC delivering intracellularly derived peptides, and class II MHC delivering extracellularly derived peptides) to the surface of the infected cell. The two classes of MHC molecules vary with respect to the length of peptides that they are able to present. The binding pocket of MHC class I molecules is blocked at either end, thereby imposing severe restrictions on the size of peptides it can accommodate (typically about 8-10 residues). The binding groove of the MHC class II molecules, on the other hand, allows peptides to protrude from the ends, and consequently much longer peptides (e.g., from about 8-30 residues) can bind. Rudensky, et al. Nature, vol. 353:622-27; Miyazaki, et al., Cell, vol. 84:531-41; Zhong, et al., J. Exp. Med., vol. 284:2061-66.
A naturally occurring MHC class I molecule comprises a glycosylated heavy chain non-covalently associated with β2-microglobulin. The class I heavy chain contains an extracellular portion having three domains termed α1 (the N-terminal-most domain), α2, and α3, a transmembrane domain of about 25 amino acid residues, and a cytoplasmic tail comprising 30-40 residues. The extracellular domain is glycosylated, with glycosylation varying depending upon species and haplotype. Each of the three α domains is about 90 residues in length. X-ray crystallographic studies of the extracellular domains reveal a platform of eight anti-parallel β strands supporting the helices in α1 and α2 domains (one α-helix in each domain) arranged in an anti-parallel fashion. A long groove separates these α-helices, which is believed to be binding site for processed antigen. The α2 and α3 heavy chain domains each contain intradomain disulphide bonds. The α3 domain is structurally similar to immunoglobulin C domains. β2-microglobulin has the structure of an immunoglobulin constant region domain, and it is essential for cell surface expression of MHC class I molecules.
In contrast, the MHC class II molecules are heterodimers of heavy (α) and light (β) chains encoded by the class genes of MHC (genes A and E in the mouse, and DP, DQ, and DR in humans), with the peptide binding groove being located between the two chains. The heavy chains vary in molecular weight between 30-34 kDa, whereas the light chains have a molecular weight of about 26-29 kDa, with much of this weight difference being due to differences in glycosylation. Despite this, the α and β chains have the same overall structure, with each comprising an extracellular portion having two domains, α1 and α2 in the α chain and β1 and β2 in the β chain, followed by short transmembrane region of about 30 residues and small cytoplasmic domain of 10-15 amino acid residues. The α2 and β2 domains are similar to the class I α3 domain and β2-microglobulin. The β1 domain contains a disulphide bond. The α1, α2, and β1 domains are N-glycosylated, whereas the β2 domain, which contains a CD4 binding site, is not. Class II molecules on APCs interact with CD4 molecules on T cells in an analogous way that that class I molecules interact with CD8 molecules. CD4 and CD8 are important in antigen presentation, which are involved in kinase recruitment that signal T cell activation.
Although the structures of class I and II molecules are similar, the groove in class II molecules is more open, and thus can accommodate longer peptides than the binding groove of class II molecules. For both class I and II molecules, the topology of the peptide-binding groove depends in part on the amino acids that comprise the groove, and peptide binding depends on the nature of the peptide's side chains and their complementarity with the binding groove. Garboczi, et al., Nature, vol. 384: 134-41; Ward and Quadri, Curr Op Immunol., vol. 9:97-106; Garcia, et al., Science, vol. 279:1166-72).
D. Antigen Processing
Antigens recognized by T cells in the context of an MHC molecule are degraded or processed so that the determinant recognized by the TCR is only a small fragment of the original antigen. Antigens are processed into peptide fragments before association with MHC molecules. TCRs are sensitive to peptides in the MHC groove, rather than the conformational determinant recognized by antibodies.
Peptides bound to MHC class I molecules are recognized by CD8+ T cells (cytotoxic T cells), and those bound to MHC class II molecules are recognized by CD4+ T cells (helper T cells). Two functional subsets of T cells can thereby be activated to initiate the destruction of cells that present, or express, the particular antigen on its surface, and thereby eliminate the source of the antigen (e.g., a pathogen or diseased cell). CD4+ T cells may also help to activate B cells, and in turn give rise to the stimulation of antibody production against the antigenic motifs of extracellular pathogens.
Infectious agents or other “non-self” antigens can reside in various intracellular compartments. Viruses and certain bacteria replicate in the cytosol or in the contiguous nuclear compartment, while many pathogenic bacteria and some eukaryotic parasites replicate in the endosomes and lysosomes that form part of the vesicular system. The immune system has different strategies for eliminating such agents from these two sites. Cells containing viruses or bacteria located in the cytosol are eliminated by cytotoxic T cells that express the cell-surface molecule CD8 and present antigens on MHC class I molecules. The function of CD8+ T cells is to kill infected cells.
Immunogenic agents located in the vesicular compartments of cells are detected by a different subset of T cells, distinguished by cell surface expression of the molecule CD4. CD4+ T cells are specialized to activate/modulate other cells and fall into at least two primary functional classes: Th1 cells, which activate various immune competent cells to destroy the intravesicular non-self antigenic agents they harbor; and Th2 cells, which help to activate B cells to, among other things, make antibodies against such antigenic agents.
To produce an appropriate response to infectious microorganisms, T cells must distinguish self from non-self (e.g., foreign) material coming from the different processing pathways. This is achieved by delivering peptides to the cell surface from these intracellular compartments using either MHC class I or II molecules. As noted above, MHC class I molecules deliver peptides that come from proteins synthesized in the cell, with the antigen being expressed in association with an MHC class I molecules (antigen:MHC complex) that can be recognized by CD8+ T cells specific for the particular antigen:MHC complex. In contrast, MHC class II molecules deliver “non-self” peptides derived from proteins that have been internalized by the cell (and are thus extracellular from the perspective of the antigen-presenting cell) and processed for presentation at the cell surface in the context of an MHC class II molecule. CD4+ T cells specific for the particular antigen:MHC class II molecule complex can then recognize the complex when presented on the surface of an APC.
E. Antigen Presenting Cells
A wide variety of cells can present antigens. In lymphoid organs, the three primary APC types are dendritic cells, macrophages, and B cells. Dendritic cells are abundant in T cell areas of lymph nodes and spleen, and are very effective in initially activiating naïve T cells. When naïve T cells encounter a specific antigen for the first time on the surface of an antigen-presenting cell (APC) in the context of an appropriate MHC molecule (i.e., priming), they are activated to proliferate and differentiate into cells capable of contributing to the removal of the particular antigen and its source (e.g., an infecting pathogen). The APCs are specialized in that they express surface molecules that synergize with a specific antigen in the activation of naïve T cells. APCs become concentrated in the peripheral lymphoid organs, to which they migrate after trapping antigen while circulating in the periphery. APCs present peptide fragments to recirculating T cells, some of which may be naïve. Dendritic cells are known to present processed antigens to macrophages, which are involved in the phagocytosis of cells in a first line of defense against infectious agents. Dendritic cells are also able to present internalized, processed antigens on both class I and class II MHC molecules. APCs are also known to be activated by armed effector T cells. B cells also serve as APCs under some circumstances.
One of the features of APCs is the expression of co-stimulatory molecules, including the potent B7 molecules, including B7-1 (CD80) and B7-2 (CD86). B7 are constitutively expressed on the surface of dendritic cells, and can be upregulated on monocytes, B cells, and other APCs. They are ligands for CD28, and its homologue CTLA-4 (CD 152), which is expressed after T cell activation. CD28 the primary co-stimulatory ligand expressed on naïve T cells. Anderson, et al., J. Immunol., vol. 159:4:1669-75. CD28 stimulation has been shown to prolong and augment cytokine production, and may be important in preventing induction of tolerance.
The activation of T cells by APCs leads to proliferation of the activated T cells and to the differentiation of their progeny into armed effector T cells. The proliferation and differentiation of T cells depends on the production of cytokines (such as the T cell growth factor, IL-2) and their binding to high-affinity receptors on the activated T cell. T cells whose TCRs are bound to antigens in the absence of co-stimulatory molecules may fail to make cytokines and instead may become anergic. This dual requirement for both receptor/antigenic interaction and co-stimulation helps to further mediate naïve T cell response.
Proliferating T cells develop into armed effector T cells. Once an expanded clone of T cells achieves effector function, the T cell clone progeny can act on any target cell that displays or expresses a specific antigen on its surface. Effector T cells can mediate a variety of functions. The killing of infected cells by CD8+ cytotoxic T cells and the activation of professional APC by Th1 cells together make up cell-mediated immunity. The activation of B cells by both Th2 and Th1 cells helps to produce different types of antibodies, thus driving the humoral immune response. Kirberg, et al., J. Exp. Med., vol. 186:8:1269-75.
F. T Cell Activation
T cells generally become sensitized to antigens by becoming trapped in lymphoid organs as the T cells drain into lymph nodes through which they circulate. Antigens introduced directly into the bloodstream, or that reach the bloodstream from an infected lymph node, are picked up by APCs in the spleen, for example, where lymphoid cell sensitization occurs in the splenic white pulp. The trapping of antigen by APCs that migrate to these lymphoid tissues combined with the continuous recirculation of T cells through the tissues ensures that rare antigen-specific T cells will encounter their specific antigen being presented by an APC. Overall, T cell activation has four stages: adhesion, antigen-specific activation, co-stimulation, and cytokine signaling.
The recirculation of naïve T cells through the lymphoid organs is orchestrated by non-specific, rapidly reversible adhesive interactions between lymphocytes and endothelial cells. Naïve T cells enter the lymphoid organs through a process that is thought to occur in a number of steps. The first step in this process is mediated by selectins expressed on the T cell. For example, L-selectin on naïve T cells binds to sulfated carbohydrates on the vascular addressins GlyCAM-1 and CD34. CD34 is expressed on endothelial cells in many tissues but is properly glycosylated for L-selectin binding only on the high endothelial venule cells of lymph nodes. L-selectin binding promotes a rolling interaction, which is critical to the selectivity of naïve lymphocyte homing. Although this interaction is too weak to promote extravasation, it is essential for the initiation of the stronger interactions that then follow between the T cell and the high endothelium, which are mediated by molecules with a relatively broad tissue distribution. (Finger, et al., Nature, Vol. 379:266-9).
Stimulation by locally bound chemokines activates the adhesion molecule LFA-1 on the T cell, increasing its affinity for ICAM-2, which is expressed constitutively on all endothelial cells, and ICAM-1, which, in the absence of inflammation, is expressed only on the high endothelial venule cells of peripheral lymphoid tissues. The binding of LFA-1 to its ligands, ICAM-1, ICAM-2, and ICAM-3, plays a major role in T cell adhesion to and migration through the wall of the blood vessel into the lymph nodes. Bachmann, et al., Immunity, vol. 7:549-57.
The high endothelial venules are located in the lymph nodes. This area is inhabited by dendritic cells, which have recently migrated from the periphery. The migrating T cells scan the surface of these APCs for specific antigen:MHC complexes. If they do not recognize antigen presented by these cells, they eventually leave the node via an efferent lymphatic vessel, which returns them to the blood so that they can recirculate through other lymph nodes. Rarely, a naïve T cell recognizes its specific antigen:MHC complex on the surface of an APC, which then signals the activation of LFA-1, causing the T cell to adhere strongly to the APC. Binding to the antigen:MHC complex also activates the cell to proliferate and differentiate, resulting in the production of armed, antigen-specific T cells. The number of T cells that interact with each APC in lymph nodes is very high, as can be seen by the rapid trapping of antigen-specific T cells in a single lymph node containing antigen.
G. Identification and Isolation of Antigen-Specific T Cells
In addition to their role in combating infections, T cells have also been implicated in the destruction of cancerous cells. Autoimmune disorders have also been linked to antigen-specific T cell attack against various parts of the body. One of the major problems hampering the understanding of and intervention on the mechanisms involved in these disorders is the difficulty in identifying T cells specific for the antigen to be studied. Accordingly, it is of great interest to be able to identify antigen-specific T cells. Additionally, it would be of great therapeutic benefit if T cells specific for a particular antigen could be (i) enriched and then reintroduced in a disease situation, (ii) selectively depleted in the case of an autoimmune disorder, or (iii) modified to alter their functional and/or phenotypic characteristics. Thus, identification and isolation of antigen-specific T cells is an essential requirement in immunology and medicine to understand and modulate immune responses.
Identification of antigen-specific T cell populations is generally accomplished by indirect means in animal models, such as by evaluating membrane markers correlated to activation or maturation of these cells. This has usually been accomplished using transgenic systems (Ignatowicz, et al., Cell, vol. 84:521-29; Sebzda, et al., Science, vol. 263:1615-18; Jameson, et al., Ann. Rev. Immunol., vol. 13:93-126). Analysis is generally done by means of flow cytometry, where a detector on a machine is capable of identifying cells bound to fluorescent substrates, such as fluoresceinated antibodies. Positively identified cells can be sorted for further use. Quantitation and isolation of antigen-specific T cells is usually accomplished by limiting dilution and cloning techniques. When using sorted cells, these approaches become quite cumbersome and are sometimes inaccurate, since the biological effects of antigen recognition can spread beyond the cells recognizing the antigen. For instance, upon engagement of the specific MHC:antigenic peptide complex, T cells produce cytokines that can affect expression of the same markers of activation in non-specific bystander T cells. Hence, in order to isolate and characterize cells with specificity for a given antigen, alternative procedures, such as T cell cloning, need to be applied. These techniques often require many months of technical procedures before results can be obtained. The rate of success, in particular for human systems, is quite low, and the population selected may not necessarily represent the biologically relevant component of the immune response to a given peptide. The direct interaction of a specific T cell with the antigen:MHC complex would thus be a preferred basis for T cell isolation.
H. Distinctions
Kendrick, et al., U.S. Pat. No. 5,595,881, discuss a method for the detection and isolation of MHC:antigen-restricted T cells. That method is performed by preparing a MHC:antigen complex isolated using metal chelating technology. The complex is then bound to a planar solid support (i.e., a glass coverslip), followed in turn by combining the immobilized complex with a biological sample so that the MHC:antigen complex may bind to and retain antigen-specific T cells. Determination of the presence of reactive MHC:antigen complexes is carried out by detecting cell proliferation.
The method of Kendrick, et al. substantially differs from the current invention. First, the MHC components of the complexes discussed in the Kendrick, et al. disclosure are immobilized on a planar solid support. In contrast, the MHC components of the current invention are not bound to a solid planar support. Indeed, in certain preferred embodiments of the instant invention, the MHC components are “free floating,” i.e., they are capable of laterally diffusing, within the fluid lipid bilayer of a liposome membrane, preferably one comprised of phosphotidylcholine and cholesterol components, as described in greater detail below. This difference is substantial in that the MHC:antigen complexes discussed in the Kendrick, et al. disclosure are believed not to be able to participate in the migration or concentration of such complexes in “capping,” which is important to improved binding and activation of bound T cells. Second, the method discussed in the Kendrick, et al. disclosure concerns detecting “natural” APCs specific for pre-selected antigen-specific T cells. Reportedly, this is accomplished by isolating antigen-specific T cells by first performing a series of steps including binding antigen via a metal chelating process to a solid support, using the antigen to capture antigen-specific MHC components, and then isolating the MHC:antigen complexes which are in turn bound to a planar solid support via a linker.
The current invention is much more versatile. It is not concerned with detecting natural APCs; instead, it concerns the isolation and manipulation of antigen-specific T cells. The manipulation of such T cells can be carried out for numerous reasons, such as to directly impact T cell function by modulating a T cell response. Such manipulations can be performed in vivo, in a column format (where artificial APCs are bound to a solid support, for example), or in solution, for example, via flow cytometry techniques such as FACS (fluorescence-activated cell sorting). The compositions of the current invention can modulate T cell function by including various functional molecules into the APC. For example, in one embodiment of the current invention, known MHC molecules may be incorporated into liposomes along with a labeled antigenic peptide for which such MHC has specificity (e.g., in the case of FACS, a biotinylated antigen). The liposome:MHC:biotinylated antigen complex may be used to bind to antigen-specific T cells. Binding can be visualized by FACS, followed by sorting the bound cells. Thus, no cell proliferation assay is necessary to identify and isolate antigen-specific T cells.
In addition to the MHC:antigen complex, the artificial APCs of this invention that are used to capture antigen-specific T cells preferably include accessory molecules to help stabilize the MHC:antigen:TCR interaction, and may also include functional molecules such as co-stimulatory molecules which, in some embodiments, may be used to activate T cells; adhesion molecules, which may be used to bind cells destined for a certain area of the body; targeting molecules that target the aAPC to a specific cell or tissue type in a patient's body; and other accessory or functional molecules such as cytokines or antibodies to cytokine receptors, which are known to have immunomodulatory effects upon T cells. Moreover, in some embodiments the current invention further provides for properly orienting these molecules in a liposome-based aAPC through the use of a novel anchoring mechanism, a preferred example of which comprises a GM-1 ganglioside molecule and the β subunit of cholera toxin. In this context, the protein of interest may be connected to a cholera toxin subunit as a fusion protein or by use of a linking moiety. By attaching the cholera toxin subunit to the molecule of interest, the cholera toxin may be bound by the GM-1 ganglioside molecule incorporated into lipid membrane of the liposome, as the GM-1 ganglioside molecule has affinity for the nonpolar region of the membrane.
In liposome-based embodiments of the invention, all of these molecules may be incorporated into the lipid bilayer of the liposome. Given the fluidity of the membrane, the molecules embedded therein can laterally diffuse and become concentrated over time, for example, at an aAPC-T cell interface, to form a structure analogous to the immunological synapse that forms between T cells and APCs. Further, other molecules may be included in the compositions that do not influence the modulation of T cell responses. Examples of such molecules include proteins useful in anchoring the artificial APC to a solid support, molecules for orienting other molecules to be included in an aAPC, as well as proteins or other molecules that target an aAPC to a cell or tissue within a patient's body. As used herein, such molecules that are not associated with modulation or T cell binding are termed “irrelevant” molecules.
Additionally, a label may be attached (covalently or non-covalently) to the antigen, an irrelevant molecule, or another aAPC component, for example, to a lipid within a liposome. The artificial APCs of the invention also allow for optional expansion of T cell populations specific for the MHC:antigen complexes using solution-based (e.g., roller bottle or bioreactor) cell culture.
The current invention represents a substantial and heretofore unrecognized advance in the MHC:antigen complex—T cell-binding art in that the artificial APC is not restricted to complexes of MHC:antigen alone or bound to a planar surface. The importance of the structural differences can not be over emphasized. The addition of accessory molecules, as well as co-stimulatory molecules, and other proteins in proper orientation (particularly in embodiments that employ liposomes) allow for substantially improved binding association and manipulation of T cells, which is very important in the identification and stimulation of antigen-specific T cells. This is especially true in solution-based FACS analysis where the functionality of antigen-specific T cells can be interpreted directly. For example, prior studies (Watts, T. H., Annals of the New York Academy of Sciences vol. 81:7564-7568.) respecting the modulation of T cells may be erroneous. There, it was demonstrated that planar membranes containing purified MHC loaded with antigen fused to glass cover slips elicited IL-2 production by T cells through the interaction of the T cell with the MHC:antigen complex. It was also shown that the same complex when formed in unilamellar vesicles (i.e., liposomes) elicited no response. Contrary to such teaching, the instant invention is based in part on the discovery that liposome vesicles containing MHC:antigen complexes can in fact elicit strong responses when combined with accessory molecules such as LFA-1, and other molecules such as co-stimulatory and adhesion molecules. We based our theory that liposomes could function without use of a planar array on the observation (by the same study cited immediately above) that crude membrane preparations of cellular material from which the MHC was purified were effective in eliciting T cell responses in both planar and vesicular forms. Subsequently, we discovered that “extraneous” matter existing in cell extracts that might be hypothesized to impart functionality to vesicular forms of lipid bilayers (as opposed to unilamellar liposomes alone) are not important to T cell binding and response. Rather, T cell binding and response is possible using vesicular forms of liposomes containing specific molecules applied in combination with liposomes (e.g., accessory molecules, co-stimulatory molecules, and adhesion molecules).
Prior research has also been inconclusive respecting the use of MHC molecules. For example, it has been shown (Buus, S., Cell, vol. 47:1071-1077) that a particular antigenic peptide binds solely to the alpha chain of the class II MHC IAd molecule, while other investigations have shown that binding interactions between T cell receptors and MHC:antigen ternary complexes use whole MHC, not just single chains of the MHC, to determine peptide sequence motifs. Exactly how much of a MHC:antigen complex must be present is not absolutely known and may vary with T cell specificity. The instant invention preferably uses either whole MHC molecules or those parts of the α and β subunits of class I and class II MHC needed to form peptide-binding cleft regions.
The current invention's use of co-stimulatory, adhesion, and other accessory molecules in a “free floating” format (in its liposome embodiments) also helps to both anchor and direct the interaction between MHC:antigen:accessory molecule and T cell receptors by providing a structure that mimics those found in the natural state. Specifically, in the liposomes used in the invention, the MHC:antigen:accessory molecule complexes in conjunction with other functional molecules are able to migrate in proper orientation in the lipid bilayer of the liposome. Such fluidity allows various components within the membrane, which may be randomly distributed prior to interaction with a T cell specific for the particular antigen being presented, to rapidly become concentrated at the T cell-liposome interface in a manner analogous to that which occurs between a T cell and an APC in situ during the formation of an immunological synapse. In preferred embodiments, the liposomes of the invention comprise preferred combinations of lipids and surfactant molecules, particularly phosphotidylcholine and cholesterol. Such aAPC embodiments provide protein presentation characteristics and protein migration properties similar to those of naturally occurring APCs, and allow the MHC:antigen complexes to easily migrate to T cell binding loci similar to “capping” events seen in natural APCs. Moreover, as representatively illustrated in the figures, the structure of the liposome-based aAPCs on the invention allows for specific “capping” of the liposomes on the surface of the T cells to which the liposomes are bound. Additionally, interaction between the T cell and aAPC-associated molecules is further enhanced by the molecules being oriented in the lipid membrane such that their active sites are positioned facing outward on the APC. Without such orientation, the ratio of properly oriented molecules to improperly oriented molecules would be expected to be around 50:50. This ratio is greatly increased using MHC, functional, and accessory proteins that have attached thereto (either by fusion protein construction or by use of a linker) an orienting moiety, a preferred example of which is cholera toxin β subunit placed in relation to the active center of the protein of interest such that upon the β subunit being bound by a GM-1 ganglioside molecule incorporated in the lipid layer of the aAPC, the protein of interest will be oriented on the outer surface of the APC, as opposed to being placed in the lumen (i.e., the internal volume defined by the liposome) of the aAPC.
Additional versatility is available with the current invention in that the artificial APCs may incorporate irrelevant molecules to be used, for example, in conjunction with separate solid support-based capture moieties for capturing generic target motifs such as irrelevant molecules. Because of the capacity for the functional molecules to migrate in the liposome, in such embodiments the irrelevant molecules can be used to anchor the aAPCs to a solid substrate (e.g., a column matrix useful for column chromatography) in a manner that will not interfere with binding of T cells having TCRs specific for the MHC: antigen complex carried by the aAPCs. As will be appreciated, such a system avoids the need for manufacturing specialized solid phase capture substrates for each antigen-specific complex.
With regard to the capture of an aAPC by a solid phase component (e.g., a column matrix having one member of a binding pair bound thereto), the target molecules used in the aAPC for binding to capture molecules (e.g., the other member of the particular binding pair) of the solid support are called “irrelevant” molecules because they do not impact the aAPC:T cell interaction. Such a design further preserves the ability of the other molecules present in the aAPC to participate in T cell capping or activation. In liposome-based embodiments, the complexes involved in such activities can diffuse away from the irrelevant molecules involved in column binding. With regard to aAPC embodiments that do not provide membrane fluidity, it is preferred that the irrelevant molecules be segregated to one region of the structure, with the complexes intended for T cell interaction being disposed in a different region.
It has been recognized that the number of receptors on a T cell is variable (Rothenberg, E., Science, vol. 273:78-79). It is also known that the number of TCRs and combination of co-stimulatory molecules and accessory molecules varies with the maturation of the T cell (Dubey, C., J. Immunol., vol. 157:3820-3289). How many such receptors are needed in any situation to elicit a T cell response is unknown. Moreover, it is known that presence of a co-stimulatory signal decreases the number of receptors necessary to activate a T cell (Viola, A. and Lanzavecchia, A., Science, vol. 273:104-106). That said, because the instant invention involves the preparation of aAPCs of defined compositions, it is now possible to control the number of MHC:antigen:accessory molecule complexes relative to other molecules, including functional molecules such as co-stimulatory and adhesion molecules, present in a given aAPC preparation. Accordingly, the binding and modulation of the T cell response at different stages of cell maturation may be “fine tuned” using the instant invention.
In another system, Nag, et al. (U.S. Pat. No. 5,734,023) disclosed MHC subunits that were complexed with antigenic peptides and “effector” molecules wherein such complexes were used to identify T cell populations that were associated with autoimmune diseases. The complexes were reportedly used to destroy and anergize such T cell populations from a patient's blood cell population. Therein, the effector molecules were disclosed as being toxins, radiolabels, etc. which may be conjugated to the MHC or antigen portion of the complexes to effecutate the identification, removal, anergy, or death of such T cell populations. Such effector molecules were not disclosed as being related to the attractive binding interactions or T cell responses to effectuate a phenotypic change in the cells. Instead, they were merely designed and intended to aid in the recognition and/or destruction of specific T cell populations. Additionally, the Nag, et al. disclosure uses lipids in the construction of micelles that are designed for intravenous injection as therapeutics.
The use of negatively charged acidic phospholipids (such as phosphatidylserine) and the lack of cholesterol or GM-1 ganglioside molecules and cholera toxin subunit in the design of such micelles differs from that of the current invention in substantial ways. For example, preferred embodiments of the instant invention use neutrally charged phospholipids such as phosphotidylcholine (Pc). It has been discovered that the aAPCs of the instant invention have substantially increased stability because of the Pc and cholesterol in environments where IL-1 is present. IL-1 is known to interact with charged phospholipids and to destabilize liposome structure. Likewise, in environments where TNF is present, the permeability of liposomes comprised of charged phospholipids (e.g., phosphotidylserine) is greatly affected. In the same manner, environments where RNase is present may also affect charged phospholipid liposome structures. In the present invention, liposome-based embodiments avoid such disruptive effects through the use of neutral phospholipids.
Additionally, the Nag, et al. disclosure provides no insight with regard to the inclusion of co-stimulatory molecules, adhesion molecules, or protein orientation mechanisms (such as the binding of cholera toxin by GM-1 ganglioside), etc.
In yet another recent disclosure, Spack, et al. (U.S. Pat. No. 5,750,356) report a method for monitoring T cell reactivity using a modified ELISPOT assay which detects various factors produced by the stimulation of T cells with numerous factors in the presence of natural antigen presenting cells. The current invention is distinguishable from the Spack, et al. disclosure in that the current invention uses artificial antigen presenting cells that, in addition to peptide loaded MHC complexes, include one more of various accessory, orienting, co-stimulatory, adhesion, cytokine, and/or chemokine molecules that can effect binding and/or modulation of T cell responses. Additionally, in embodiments that require solid support binding, the aAPCs of the invention include irrelevant molecules.
In still another disclosure, Wilson, et al. (U.S. Pat. No. 5,776,487) disclose the use of liposome structures for detecting analyte in a test sample, wherein the liposome contains only an analyte-specific ligand and a haptenated component used to bind to a receptor moiety on a solid phase, thereby allowing capture of a test analyte on a solid support for detection. Thus, it is vastly divergent from the concept of the current invention.
The present invention radically advances the art by providing synthetic compositions that enable, among other things, the stimulation and modulation of the activity of antigen-specific T cells. This has been accomplished by providing structures (e.g., liposomes and dendrimers) that allow high local concentrations of the molecules necessary to effect modulation of T cell activity to become localized at an aAPC-T cell interface and thereby facilitate binding and T cell capping. Capping is the phenomenon by which the T cell focuses the relevant molecules to the portion of the cell surface where binding has occurred, thus amplifying the binding, and subsequently the signaling of the event to the cell's other components.